Effect of light on ascorbic acid biosynthesis and bioinformatics analysis of related genes in Chinese chives

Ascorbic acid (AsA) is an essential nutritional component and powerful antioxidant in vegetables, and in plants, AsA levels are regulated by light. AsA levels in the leaves of Chinese chive (Allium tuberosum Rottler ex Spr), a popular vegetable, are poorly understood. Thus, this study was performed to assess the influence of light on AsA biosynthesis in chive and select related genes (AtuGGP1 and AtuGME1); in addition, bioinformatic analyses and gene expression level assays were performed. The biological information obtained for AtuGGP1 and AtuGME1 was analysed with several tools, including NCBI, DNAMAN, and MEGA11. After different light treatments were performed, the Chive AsA content and AtuGGP1 and AtuGME1 expression levels were determined. These results suggest that 1) compared with natural light, continuous darkness inhibited AsA synthesis in chives. 2) The amino acid sequences of AtuGGP1 and AtuGME1 are very similar to those of other plants. 3) The trends observed for the expression levels of AtuGGP1 and AtuGME1 were consistent with the AsA content observed in chives. Hence, we speculated that light controls AsA biosynthesis in chives by regulating AtuGGP1 and AtuGME1 expression. This study provided impactful and informative evidence regarding the functions of GGP and GME in chives.


Introduction
Chinese chive (Allium tuberosum Rottler.ex Spr.), a perennial plant of the Allium genus, has been widely cultivated and naturalized worldwide [1].Due to their high nutritional and medicinal properties, Chinese chives have become increasingly popular in food inventories as multifunctional vegetables; this popularity greatly reflects consumers' awareness of the medicinal potential of chives to improve health and prevent chronic diseases [2][3][4][5].
Ascorbic acid (AsA), also known as vitamin C (VC), is a potent water-soluble antioxidant in humans and plays important roles in the synthesis of organic components within the extracellular matrix [6].In plants, AsA acts as a major antioxidant and enzymatic cofactor that participates in several cellular and molecular processes [7,8].Several pathways for AsA biosynthesis have been identified, including the D-mannose/L-galactose pathway [9], D-galacturonate pathway [10], myoinositol pathway [11], and L-gulose pathway [12].The L-galactose pathway has been identified as the primary pathway for AsA biosynthesis in many higher plants [13][14][15][16][17]. Gene expression levels in the AsA metabolic pathway are closely related to changes in AsA accumulation [18].Several key genes involved in AsA accumulation have been identified in various plants.In this complex process, GDP-L-galactose phosphorylase (GGP) is a rate-limiting enzyme that plays a vital role in AsA synthesis and is a major determinant of AsA concentrations [19].GGP is the first specific enzyme in the L-galactose pathway and catalyses the conversion of GDP-L-galactose into L-galactose-1-phosphate [20].Additionally, GDP-mannose 3,5-epimerase (GME) synergistically controls AsA biosynthesis [21] and catalyses the epimerization of GDP-D-mannose, generating GDP-L-galactose in the L-galactose pathway and GDP-L-gulose in an alternative L-gulose pathway [22,23].The AsA content was increased by 1.5-to 2.5-fold by the overexpression of GME and GGP genes in the D-mannose/ L-galactose pathway within Arabidopsis and rice leaves, which was achieved through the genetic manipulation of the AsA biosynthesis process [24,25].Previous studies revealed that the AsA content decreased significantly in GME-silenced tomatoes [26].Among the GGP deletion mutants, the AsA content in tomato fruit was significantly reduced [27].
Light is essential for the activation of AsA biosynthesis [28,29], and research has shown that light induces many genes in the AsA synthesis pathway (such as GMP, GME, GGP, GPP, GalDH, and GLDH) that contain optical response elements on their promoters [30].Under dark conditions, the transcription levels of GGP and GME were decreased, and the AsA content in Arabidopsis leaves decreased by 91%; however, after prolonged illumination, the AsA content was 1.71 times greater than that in the control [28].Shading leads to a decrease in the expression level of most genes that synthesize AsA, of which GME and GGP are the most reduced [31].Compared with the conventional optical period (12 h/12 h), continuous light (24 h/0 h) significantly increased the content of AsA in lettuce leaves [32].Continuous light also gradually upregulated the expression of GME and GGP genes [28].
Here, we show that plant GGP and GME expression levels are largely influenced by light, which thus affects AsA biosynthesis.However, studies on how light affects AsA synthesis and GGP and GME expression in chives are scarce.Therefore, this study was performed to determine the physicochemical properties of GGP and GME in chive by bioinformatic analysis, as well as the influence of light on the AsA content and GGP and GME expression levels in chives, to provide more impactful and informative information on the functions of GGP and GME in chive.

Plant materials
Seeds of the A. tuberosum cultivar '791' were germinated on substrates (peat: vermiculite = 3: 1) in a balcony greenhouse located in the Beijing Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences (latitude: 39˚56 0 18 00 N; longitude: 116˚17 0 03 00 E).After the first crop harvest, the chives were treated as follows: 1) the plants were grown under natural light (L) or continuous darkness (D).Fresh leaves of plants were collected after 10 days, 20 days, 30 days and 40 days under treatments (samples were used in three replicates)., and fresh leaves were collected (samples were used in three replicates).The plants in each treatment group were irrigated with nutrient solution every 3 days.The composition was as follows: Ca(NO 3 ) 2 1.0 mM, KNO 3 4.0 mM, NH 4 NO 3 2.0 mM, KH 2 PO 4 2.0 mM, (NH 4 ) 2 SO 4 1.0 mM, MgSO 4 �7H 2 O 1.0 mM [33].All the collected samples were immediately frozen in liquid nitrogen and stored at -80˚C before use.

Total RNA isolation and first-strand cDNA synthesis
Total RNA from the chive leaves was extracted using a Vazyme FastPure Universal Plant Total RNA Isolation Kit (Nanjing Novizan Biotechnology Co., Ltd.) in accordance with the manufacturer's instructions.Total RNA was dissolved in 100 μL of RNase-free water, and the concentrations were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).cDNA was synthesized using the TRAN EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing All-style Gold Biotechnology Co., Ltd.).Briefly, total RNA was mixed with dNTPs in a total volume of 20 μL and incubated for 5 min at 65˚C, 30 min at 42˚C, or 5 s at 85˚C in accordance with the manufacturer's instructions.First-strand cDNA was stored at -20˚C before use.

Cloning the full-length cDNA of AtuGGP1 and AtuGME1
The transcriptome sequencing results revealed one homologous GGP gene sequence and one homologous GME gene sequence, which contained complete open reading frames (ORFs).We named them AtuGGP1 (387 bp) and AtuGME1 (1140 bp).The specific primers AtuGGP1-FP/ AtuGGP1-RP and AtuGME1-FP/AtuGME1-RP were designed for these two genes (Table 1) using SnapGene 7.0.2.0 to amplify the cDNA sequences of AtuGGP1 and AtuGME1.Firststrand cDNA was used as a template to amplify the cDNA of AtuGGP1 and AtuGME1 with 2×A8 PCR Mix (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's instructions.The PCR products were purified, cloned and inserted into the pEASY 1 -Blunt E2 vector (TransGen Biotech, Beijing, China), and sequenced by Shanghai Sangon Biotech Co., Ltd.(Shanghai, China).

Bioinformatic analysis
The protein physicochemical properties were analysed by ProtPatham, and the secondary structures were predicted by NovoPro.Both ProtPatham and NovoPro were used to analyse the protein hydrophilicity.The Molecular Bioinformatics Center was used to predict protein subcellular localization.The protein phosphorylation sites were predicted using NetPhos 3.1.Finally, amino acid homologous sequence alignment and phylogenetic tree construction were completed using NCBI BLAST, DNAMAN and MEGA11 (Table 2).

Analyses of AtuGGP1 and AtuGME1 expression by real-time quantitative PCR
Real-time quantitative PCR cDNA amplification was carried out on a Bio-Rad CFX Opus 96 real-time PCR system (CFX Opus 96, USA).SYBR Green Mix (2×) (Toyobo (Shanghai)

AtuGME1-FP ATGGGTGATAACTCGGACAAAAC
AtuGME1-RP TTAGAGCCCTTCATTTCCATCAG https://doi.org/10.1371/journal.pone.0307527.t001 Biotech Co., Ltd.) was used in all reactions according to the protocol described by the manufacturer.Gene-specific primers were designed using Primer Premier 5.0 software (Table 3).The volume of each reaction was 20 μL (10.0 μL of SYBR Green Mix (2×), 0.5 μL of forwards and reverse primer each, 2.0 μL of cDNA, and 7.0 μL of RNase-free ddH 2 O).AtuSKP1/ DN253_c0_g1 was selected as the housekeeping gene, and the relative expression levels were calculated using the 2 -44Ct method.Three biological replicates were performed.

Extraction and determination of AsA
Ascorbic acid oxidase (AAO) catalyses the oxidation of AsA to produce dehydroascorbic acid (DHA) [34].Thus, the AsA content can be calculated by measuring the percentage of AAO that oxidizes AsA [35].Briefly, plant leaves (0.1 g) were mixed with trichloroacetic acid (TCA) solution (1 mL) for homogenization and centrifuged at 8000 × g for 20 min.Afterwards, the supernatants were mixed with AAO.Then, the absorbances were measured after 10 s and 130 s by a spectrophotometer at a wavelength of 265 nm, and three biological and technical replicates were conducted.

Data analysis
The significance of the differences in the data were assessed by PASW Statistics 18 by making multiple comparisons, and the charts were drawn with Excel 2016 software.

Full-length cDNA cloning
With the cDNA of the leaf as the template, the ORFs of AtuGGP1 and AtuGME1 were amplified via PCR to obtain two specific fragments.Agarose electrophoresis of the PCR products revealed that AtuGGP1 had a single band at approximately 300 bp, AtuGME1 had a single band at approximately 1000 bp, and there were no nonspecific amplification bands (Fig 1).These fragments were linked with the pEASY 1 -Blunt E2 vectors, and the positive clones were selected.After sequencing and comparison were performed, the sequences of AtuGGP1 and AtuGME1 were confirmed.According to the amino acid sequence inferred from SnapGene

Bioinformatic analysis
The protein physicochemical properties of AtuGGP1 and AtuGME1 were analysed using Prot-Patham, and the results showed that the relative molecular weight of the AtuGGP1 protein was 14676.84D, the molecular formula was C 650 H 1024 N 170 O 198 S 9 , the aliphatic index was 86.09, the theoretical isoelectric point was 5.09 (<7), and the instability coefficient was 43.77 (>40), indicating that AtuGGP1 is an unstable acidic protein.The relative molecular weight of the AtuGME1 protein was 42804.56D, the molecular formula was C 1903 H 2936 N 516 O 570 S 20 , the aliphatic index was 72.59, the theoretical isoelectric point was 5.83 (<7), and the instability coefficient was 39.00 (<40), indicating that AtuGME1 is a stable acidic protein (Table 4).
The protein secondary structures of AtuGGP1 and AtuGME1 were predicted by NovoPro.The results showed that the α-helix, extended chain, and irregular curl of the AtuGGP1 protein accounted for 56.25%, 10.94% and 32.81% of the structure, respectively.The α-helix, extended chain, and irregular curl of the AtuGME1 protein accounted for 31.66%,17.41% and 50.92% of the structure, respectively (Fig 3).The protein hydrophilicities of AtuGGP1 and AtuGME1 were analysed via ProtPatham and NovoPro, and the results indicated that the maximum hydrophobicity of AtuGGP1 was 1.8 (at positions 10 and 123), the minimum hydrophobicity  was -2.9 (at position 62), and the average hydrophilicity was -0.291, suggesting that AtuGGP1 is a hydrophilic protein.The maximum hydrophobicity of AtuGME1 is 1.5 (at positions 37 and 143), the minimum hydrophobicity is -2.7 (at position 65), and the average hydrophilicity is -0.445, suggesting that AtuGME1 is a hydrophilic protein (Table 4; Fig 4).Molecular Bioinformatics Center online software predicted that the AtuGGP1 and AtuGME1 proteins are most likely located in the cytoplasm.Phosphorylation affects protein activity and is closely related to signal transmission.Net-Phos 3.1 predicted that the AtuGGP1 protein contains 10 highly reliable phosphorylation sites, including 4 3 threonine, and 3 tyrosine residues.The AtuGME1 protein contains 35 highly reliable phosphorylation sites, including 16 serine, 13 threonine, and 6 tyrosine residues (Fig 5).It is speculated that AtuGGP1 and AtuGME1 are mainly composed of serine and other amino acids that act as auxiliaries, completing phosphorylation and regulating biological functions.
The amino acid sequences encoded by AtuGGP1 and AtuGME1 were used to select homologous sequences from other plants.These results suggest that the amino acid sequences of AtuGGP1 and AtuGME1 are highly similar to those of other plants (Figs 6 and 7).The amino acid sequence of AtuGGP1 is highly homologous to that of 8 other plant species, including blue leek, pineapple, coconut, plantain, and oil palm, at over 50%.The amino acid sequence of AtuGME1 is highly homologous to that of 16 plant species, including asparagus, Calamus, Iris pallida, and millet, which are more than 90% homologous.Based on the AtuGGP1 and AtuGME1 protein sequences, phylogenetic trees were constructed using MEGA11 software.Based on the phylogenetic tree analyses, AtuGGP1 was not closely related to plants such as oil palm, coconut, and plantain, and blue leek contained the most closely related gene to AtuGGP1.The plant with the most closely related gene to AtuGME1 was asparagus, followed by calamus and Iris pallida; in contrast, plants such as Magnolia sinica, papaya and pistachio had relatively distant relationships with AtuGME1 (Fig 8).

AsA content and gene expression level
As shown in Fig 9A, the AsA content gradually decreased as the chives grew in natural light but gradually increased after 30 d.When the chives grew in the dark, the AsA content gradually decreased.On the 30th and 40th days, the chive AsA content was significantly lower than that on the 10th and 20th days.During the 40-day growth period, the AsA content was greater in the chives that grew in natural light that in the chives that grew in continuous darkness.).
These results indicate that light affects AtuGGP1 and AtuGME1 expression levels.When chives were grown in natural light, AtuGGP1 and AtuGME1 expression levels gradually decreased, but after 30 d, the levels gradually increased (Fig 10A).As the chives grew in the dark, AtuGGP1 and AtuGME1 expression levels gradually decreased (Fig 10A).Thus, the change in AsA content in chives was consistent with the changes in AtuGGP1 and AtuGME1 expression levels.Additionally, compared with those in chives growing in natural light, AtuGGP1 and AtuGME1 expression levels in chives growing in continuous darkness were  ).

Discussion
AsA is among the essential components of fruit and vegetable nutritional quality [30].Many horticultural plants contain high levels of AsA; for example, the AsA content in mature fruits of prickly pear, hawthorn, rosehip, and cherokee rose-hip can reach 800 mg per 100 g.The AsA content is usually higher in wild fruits than in cultivated fruits [36].AsA is a powerful antioxidant that may decrease the incidence of several illnesses, such as cancer and cardiovascular diseases [37]; however, the human body cannot synthesize AsA because of a lack of the L-guluronic acid-1,4-lactone oxidase enzyme [38].Thus, plant-derived foods are the main source of AsA for humans.Furthermore, AsA is an essential antioxidant in plants that scavenges reactive oxygen species (ROS) generated during exposure to biotic or abiotic stresses and those produced during normal growth and development [7,39,40].
Chinese chives are cultivated in China and other Asian countries as well as many European countries because this popular vegetable is rich in vitamins, fibre, and sulfur compounds and exhibits antibiotic properties [41,42].Although these nutritional qualities are well known, knowledge regarding AsA compounds in the leaves of Chinese chives is lacking.Light is an important signal for the activation of plant AsA biosynthesis, and researching the effects of light on AsA biosynthesis is convenient and inexpensive.Hence, we assessed the influence of light on AsA biosynthesis and selected related genes in chives; performed bioinformatic analyses; and performed assays to investigate the levels of gene expression in chive.
L-galactono-1,4-lactone dehydrogenase (GalLDH) is involved in the final step of the Lgalactose pathway and catalyses the formation of AsA from L-galactono-1,4-lactone. Previous studies revealed that the overexpression of GME in tomato plants increased the AsA content and plant tolerance to salt, drought, and low temperatures [47,48].In contrast, after two GME genes in tomatoes were deleted, the AsA content decreased, the stems became brittle, and the fruit hardness decreased [26].Tomato GGP1 is a homologous gene of Arabidopsis VTC2 and VTC5, and inhibiting GGP1 expression can reduce fruit cold resistance and yield [27].The simultaneous overexpression of GME and GGP significantly increased the AsA content in cherry fruits [49].There was a significant correlation between the expression level of the GGP gene and the AsA content in kiwifruit leaves and fruits during the growth period [14].GGP and GME play vital roles in plant AsA synthesis and significantly influence AsA content.Our results showed that the change in AsA content in chives was consistent with the changes in the expression levels of AtuGGP1 and AtuGME1 (Figs 9 and 10), which corresponds with previous studies on tomato [47] and kiwifruit [14].AtuGGP1 and AtuGME1 might strongly influence AsA biosynthesis in chives.
Light exerts a regulatory effect on AsA levels in plants by stimulating the L-galactose pathway, inducing gene expression, and increasing plant AsA content via a series of important light signal transcription factors and proteins [50].GGP and GME contain many optical response elements in their promoters [30].For example, the Rosa roxburghii GGP promoter contains photoinduction elements such as ACE, AE-box, Box4, CAG-motif, G-Box, GAmotify, GAG-motif, GTI-motif and GATA-motif.Under continuous light treatment, most rosa roxburghii GGP promoter mutants showed a significant increase in activity, but their activity decreased to varying degrees under continuous dark treatment [51].
Early studies revealed that the AsA content in Arabidopsis leaves decreased by 40% after 24 h of dark treatment [52], while under high light conditions, the AsA content in kiwifruit fruits [53], tomato leaves and fruits [54], apple fruits [55], and cowpea seedlings [56] significantly increased.Under shading, the AsA content of tomato fruits decreased by 30%, and the GME and GGP expression levels decreased concurrently [31].Our research revealed that the AsA content of chives grown in natural light was greater than that of chives grown in continuous darkness during the 40-day growth period (Fig 9A).In addition, as the lighting duration increased, the chive AsA content gradually improved (Fig 9B).The trends in the expression levels of AtuGGP1 and AtuGME1 were consistent with the abovementioned trends in AsA content (Figs 9 and 10).Therefore, we speculate that AtuGGP1 and AtuGME1 control chive AsA biosynthesis via optical response elements in their promoters.
The GGP protein plays a role in AsA synthesis and may function as a growth regulation factor.Research on Arabidopsis vtc2 mutants revealed that the VTC2:GUS fusion protein is located simultaneously in the cytoplasm and nucleus; these results suggest that GGP plays an enzymatic role in the cytoplasm and performs specific functions to regulate gene expression in the nucleus [57].Similarly, the tomato SlGGP-GFP fusion protein is localized in both the cytoplasm and nucleus of onion cells [58].Additionally, previous research has shown that plants might experience slower cell division and growth due to the inactivation of GME [59], suggesting that GME controls plant growth.In addition to AsA synthesis, the functions of GGP and GME should be further studied.
In conclusion, light exerts a regulatory effect on AsA biosynthesis in chives.GGP and GME play vital roles in AsA synthesis, as they are light-induced and control plant AsA synthesis.The amino acid sequences of AtuGGP1 and AtuGME1 are very similar to those of other plants.AsA synthesis was inhibited in chives grown in continuous darkness compared to chives grown in natural light.Moreover, the trends in AtuGGP1 and AtuGME1 expression were generally consistent with the changes in AsA content in chives.These results suggest that light controls AsA biosynthesis in chives by regulating AtuGGP1 and AtuGME1 expression.